Wound Dressings Containing Complexes of Transition Metals and Alginate for Elastase-Sequestering

ABSTRACT

Transition metal (e.g., silver and copper) derivatized phosphorylated polysaccharides (cellulose, starch, gauze) provide antimicrobial and elastase sequestration properties to wound dressings, and the wound dressing have enhanced water sorption and elastase sequestration when used with alginates. Wound dressings with alginates (e.g., silver alginate, crosslinked alginates, etc.) provide enhanced wound fluid absorption as well as elastase sequestration.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of application Ser. No. 10/446,806 filedMay 29, 2003, which is a continuation-in-part of U.S. patent applicationSer. No. 09/515,172, entitled “Wound Dressing with Protease-LoweringActivity,” filed on Feb. 29, 2000, that are incorporated herein inentirety by reference.

FIELD OF THE INVENTION

The invention generally relates to wound dressings and their methods ofuse. In particular, the invention provides wound dressings withassociated active agents such as protease inhibitors and sequestrantswhich enhance the healing of wounds, especially chronic wounds.

BACKGROUND OF THE INVENTION

The normal response to tissue injury is a timely and orderly reparativeprocess that results in sustained restoration of anatomic and functionalintegrity (Lazarus, et al. 1994). In contrast, in chronic ulcers, thehealing process is prolonged, incomplete and proceeds in anuncoordinated manner resulting in poor anatomical and functionaloutcome. Clinically, wounds are categorized as acute and chronic basedon the timeliness of healing.

Most chronic ulcers are associated with a small number of well-definedclinical entities particularly chronic venous stasis, diabetes mellitus,and pressure ulcers. These conditions are responsible collectively forapproximately 70% of all chronic ulcers (Nwomeh et al. 1998). Theincidence and prevalence of chronic ulcers vary considerably but areespecially high in spinal cord injury patients as well as the elderlyand nursing home population. As our society continues to age it ispredicted that the incidence of chronic ulcers will continue to increasedramatically. Patients with pressure ulcers also have a significantsocioeconomic impact on our society. For example, health careexpenditures for treating pressure ulcers alone have been estimated toexceed $3 billion a year (Nwomeh, et al. 1998). Normal healing involvesa complex cascade of events involving interaction among many cell types,soluble factors and matrix components. Healing can be arbitrarilydivided into overlapping temporal phases of coagulation, inflammationfibroplasia and finally remodeling. Most of the events are cytokineregulated. Normally, during the inflammatory phase, polymorphonuclearleukocytes (PMNs) are the first of the leukocytes to appear. Theyproduce various proteases such as MMP-8 (collagenase) and elastase,which help to remove damaged matrix and aid in healing. In both the openacute and chronic wound, various cytokines are important in contractionand spontaneous closure of the wound as well as angiogenesis. Undernormal circumstances, closure of the open wound is aided further byepithelization as these surface cells seal the final closure.

Chronic wounds are very different. For example, pressure ulcers arecharacterized by deep tissue necrosis with loss of muscle and fat thatis disproportionately greater then the loss of overlying skin (Falanga,et al. 1998). These defects are common among the immobilized anddebilitated. There are approximately 225,000 spinal cord injury patientsin the United States and approximately 9,000 new cases per year.Approximately 60% of these patients develop pressure ulcers and theannual cost is greater then $25,000 per patient for medically relatedcare (Allman, 1998) If the elderly nursing home population with pressureulcers in added to the spinal cord injury population then the figure forthe care of all pressure ulcers is enormous.

To date, the majority of the effort to improve rates of healing ofchronic wounds have focused on the use of exogenous peptide growthfactors and cell based products such as cytokines. For the most part,these attempts have met with little notable success. Another alternativeapproach has been the use of “skin substitutes” such as Apligraf(matrix+cells) and Dermagraft (matrix+cells). While this second approachhas shown some promise, its expense presently greatly limits its use tothe richer developed countries. Various modifications of the wounddressings have also been suggested as a means to augment would healing.

Further examples include:

U.S. Pat. No. 5,098,417 to Yamazaki et al. teaches the ionic bonding ofphysiologically active agents to cellulosic wound dressings.

U.S. Pat. No. 4,453,939 to Zimmerman et al. teaches the inclusion ofaprotonin in composition for “sealing and healing” of wounds. U.S. Pat.No. 5,807,555 to Bonte et al. teaches the inclusion of inhibition foralpha-1-protease, collagenase, and elastase in pharmaceuticalcompositions for promotion of collagen synthesis.

U.S. Pat. No. 5,696,101 to. Wu et al., teaches use of oxidized cellulose(e.g. Oxycel) as a bactericide and hemostat in treatment of wounds.

World Patent WO 98/00180 to Watt et al. teaches complexation of oxidizedcellulose with structural proteins (e.g. collagen) for chronic woundhealing; and references the utility of oligosaccharide fragmentsproduced by the breakdown of oxidized cellulose in vivo in the promotionof wound healing.

Neutrophils are a predominant infiltrating inflammatory cell typepresent in the acute inflammatory response. Neutrophils functionprimarily to destroy invading pathogens and to debride devitalizedtissue at the site of injury. The normal adult produces approximately10.sup.11 neutrophils per day. To function effectively in host defense,they must migrate to the site of inflammation and release selectively alarge repertoire of lytic enzymes, antimicrobial peptides, and potentoxidants from cytoplasmic granules. Under other conditions, theneutrophil has been implicated in causing disease by damaging normalhost tissue. Such inflammatory tissue injury are important in thepathogenesis of a variety of clinical disorders including arthritis,ischemia-reperfusion tissue injury and systemic inflammatory responsesyndrome (SIRS) and the acute respiratory distress syndrome (ARDS)(Weiss, 1989) There is strong evidence that neutrophils also may have asignificant role in the pathophysiology of pressure ulcers.

Neutrophils are a prevalent cell type in pressure ulcers (Diegelmann, etal. 1999; Paloahti. et al. 1993; Rogers et al. 1995) In addition, thereis direct evidence correlating neutrophil products with chronic pressureulcers (Yager, et al. 1996; Yager, et al. 1997). This includesneutrophil elastase, gelatinase (MMP-9) as well as collagenase (MMP-8)(Wysocki, 1996; Wysocki et al, 1993; Yager et al. 1997; Yager et al.1996). Therefore, these observations and the evidence that neutrophilshave been implicated in tissue destruction in other inflammatoryprocesses give strong credence to the hypothesis that neutrophilproducts are involved in the pathogenesis of pressure sores andsubsequent failure to heal. Neutrophil-derived MMP-8 has been shown tobe the predominant collagenase in both acute and chronic wounds (Nwomeh,et al. 1999).

Neutrophils contain large amounts of elastase (1 pg/cell). This serineprotease has a broad substrate spectrum. As with neutrophil-derivedMM-8, elastase levels have also been found to be significantly elevatedin fluid derived from pressure ulcers (Yager et al. 1997) The presenceof high levels of active elastase with a wound site may have importantimplications for wound healing therapies utilizing peptide growthfactors. Elastase present in chronic wounds can degrade peptide growthfactors such as PDGF and TGF-b (Yager et al. 1997). Moreover, cellsurface receptors for peptide growth factors may themselves befunctionally inactivated by the actions of elastase. Elastase may alsocontribute to the overall proteolytic environment of chronic wounds. Itis known to proteolytically inactivate the specific inhibitor, TissueInhibitor of Metalloproteinases (TIMP). In addition, elastase itself mayparticipate in proteolytically activating collagenase and gelatinasezymogens. Obviously, an unregulated proteolytic environment can be asignificant aspect of the pathophysiology of chronic wounds.

It would be highly beneficial to have available additional methods forenhancing wound healing. In particular, methods directed to bringing theproteolytic environment of wounds under control in order to promotewound repair would be desirable. Such methods would be useful in thetreatment of wounds in general, and chronic wounds in particular.Further, it would be highly beneficial if such methods were inexpensiveand thus widely accessible.

A number of patents and patent applications describe the use ofalginates in the treatment of burns or wounds. For example, U.S. Pat.No. 6,696,077 to Scherr describes various silver alginate foamcompositions, and U.S. Pat. No. 6,809,231 to Edwards describescross-linked alginate formulations.

A number of patents and patent applications describe the use of silverions as antimicrobial agents. For example, U.S. Patent Publication2005/010900 to Qin describes polysaccharide fibers formed with alginatethat contain a silver compound as an antimicrobial agent, and U.S.Patent Publication 2004/0241213 to Bray describes carboxymethylcellulose or alginate fibers in a mixture that contains silver ions.Neither reference describes a configuration where alginates are positionin a wound dressing for sequestration of elastase.

SUMMARY OF THE INVENTION

It is an object of this invention to provide novel wound dressings forthe treatment of wounds, especially for the treatment of chronic,non-healing wounds. The wound dressings of the instant invention arecomprised of a support matrix which is preferably cellulose,carboxymethylated cellulose, dialdehyde gauze, sulfonated gauze; andphosphorylated gauze, in combination with silver or copper, alginate orsilver alginate. The wound dressings combine antimicrobial activitytogether with elastase sequestration and provide for enhanced woundhealing in burns, surgical wounds, ulcers and chronic wounds, etc. Forexample, in some embodiments, the gauze may be derivatives with silverand then coated with alginate. In other cases, the gauze may be coatedwith silver alginate which may be bound to the support matrix or be ableto dissociate from the support matrix so as to allow migration in thewound microenvironment. When phosphorylated gauze, sulfonated gauze ordialdehyde gauze is used as a support matrix, the wound dressingbenefits from elastaste sequestration by the gauze itself as well as bythe alginate. The sequestrants bind proteases found in the wound fluidand remove them from the wound microenvironment.

The invention also provides methods of use for the wound dressings,including a method for sequestering elastase at a wound site. Thismethod comprises the step of contacting the wound site with a wounddressing selected form the group consisting of carboxymethylcellulose,dialdehyde gauze, sulfonated gauze, and phosphorylated gauze togetherwith transition metal such as silver and alginate.

The dressings may be applied to wounds in order to enhance wouldhealing, especially the healing of chronic wounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Assessment of reduction in human neutrophil elastaseactivity in samples of HLE after exposure to modified cotton gauze. 3A:HLE samples were exposed to three different oxidized cotton gauzesamples corresponding to gauze Treatment Methods 1, 2 and 3 (seeMethods, Preparation of Dialdehyde Cotton Gauze). 3B: HLE samples wereexposed to 25 and 50 mg of two different carboxymethylated cotton gauzesamples, III and IV (see Methods, Preparation of CarboxymethylatedCotton Gauze). Untreated gauze was employed as a control. Data aremean±S.D. of triplicate determinations.

FIGS. 2A-C. Reaction progress curves for gauze-treated solutions ofelastase. Substrate hydrolysis was performed with a 60 .mu.M solution ofMeOSuc-Ala-Ala-Pro-Val-pNA and reaction rates monitored byspectrophotometric measurement of the release of p-nitroaniline at 405nm. 25, 50 and 75 mg samples of phosphorylated cotton gauze (PSC, 5A),sulfonated cotton gauze (SOC, 5B) and dialdehyde cotton gauze (DAG, 5C)were compared with 75 mg of untreated cotton gauze (UT).

FIG. 3 Initial velocities (v_(o)) of residual elastase activity insamples exposed to untreated gauze (UT), dialdehyde gauze (DAG),sulfonated gauze (SOC), carboxymethylated gauze (CMC) and phosphorylatedgauze (PSC), compared to a sample that was not treated with gauze (Bk).Weights of gauze samples were 75 (A), 50 (B), and 25 {circle over (C)}mg. Data are mean±S.D. of triplicate determinations. All aresignificantly different from control, p<0.05, as determined by analysisof variance.

FIG. 4 a-f are graphs of optimization experiments show the amount ofreagent used for phosphorylation of the substrate versus thesequestering properties of the products at three different temperatures(4 a-c), and for the silver derivative of the equivalent phosphorylatedsubstrate at the same temperatures (4 d-f).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention is based upon the previously unrecognizeddiscovery that active agents such as inhibitors and sequestrants ofproteases may be used as healing accelerants of wounds, and of chronicwounds in particular. These inhibitors and sequestrants may bephysically applied on wound dressings, or in the alternative may beionically or covalently conjugated to a wound dressing material forpurposes of sustained release of active agent or sequestration ofendogenous constituents from the wound environment. In a preferredembodiment of the present invention, the active agents inhibit or bindcationic, neutrophil-derived proteases such as neutrophil elastase.

Specific pharmacological effects of proteases inhibitors and sequencesassociated with wound dressings include inhibition of the breakdown ofgrowth factors that stimulate migration of cells to the ulcer site ofthe wound, leading to the growth of new tissue that heals the openwound. This technology is broadly applicable to all forms of chronicwounds including diabetic ulcers and decubitus bedsores. Both peripheraland central administration of the compounds formulated on wounddressings accelerate wound healing of chronic wounds. Silver complexedto matrix and silver alginate are shown herein to have potent elastasesequestering properties which were heretofore previously unrecognizedand thereby inhibit proteases such as human elastase and thus preventgrowth factor and tissue degradation. Alternatively, the silver alginatemay be bound (chemically (ionic or covalently) or physically) to thewound dressing. As a component of such a matrix, they are able tosequester destructive proteases from the microenvironment of the wound,thus preventing the degradation of growth factors and fibronectin thatwould otherwise occur.

The therapeutic administration of the modified wound dressingscontaining inhibitors include a pharmacologically effective dose of theinhibitor or sequestrant (e.g., silver alginate, or silver alginate incombination with a phosporylated, sulfonated or dialdehyde gauze) whenused in the treatment of a patient in need thereof. The dose ofinhibitor or sequestrant required on the wound dressing to promoteaccelerated healing in the patient ranges from about 0.2 mg/gram fiberto about 200 mg/gram fiber per day, with this in turn being dependentupon specific factors including patient health, wound type, etc. Theterm “patient” used herein is taken to mean mammals such as sheep,horses, cattle, pigs, dogs, cats, rats, mice and primates, includinghumans.

The term “wound dressing” used herein is taken to include anypharmaceutically acceptable wound covering or support matrix such as:

a) films, including those of a semipermeable or a semi-occlusive naturesuch as polyurethane copolymers, acrylamides, acrylates, paraffin,polysaccharides, cellophane and lanolin.

b) hydrocolloids including carboxymethylcellulose protein constituentsof gelatin, pectin, and complex polysaccharides including Acacia gum,guar gum and karaya. These materials may be utilized in the form of aflexible foam or, in the alternative, formulated in polyurethane or, ina further alternative, formulated as an adhesive mass such aspolyisobutylene.

c) hydrogens such as agar, starch or propylene glycol; which typicallycontain about 80% to about 90% water and are conventionally formulatedas sheets, powders, pastes and gels in conjunction with cross-linkedpolymers such as polyethylene oxide, polyvinyl pyrollidone, acrylamide,propylene glycol.

d) foams such as polysaccharide which consist of a hydrophilicopen-celled contact surface and hydrophobic closed-cell polyurethane.

e) impregnates including pine mesh gauze, paraffin and lanolin-coatedgauze, polyethylene glycol-coated gauze, knitted viscose, rayon, andpolyester.

f) cellulose-like polysaccharide such as alginates, including calciumalginate, which may be formulated as non-woven composites of fibers orspun into woven composites.

Preferred wound dressings are polysaccharide containing support matriceswhich are derivatized with silver or copper and/or which have silveralginate bound to or placed upon them, and it is envisioned to includechitosans, alginates (e.g., cross-linked or in a form other than silveralginate) and cotton or carboxymethylated cotton in the form of gauze,films, hydrocolloide, hydrogels, hydroactives, foams, impregnates,absorptive powders and pastes,

Especially preferred wound dressings include cotton cellulose formed aswoven or non-woven gauze. This type of wound dressing has the advantageof being readily available and relatively inexpensive. In this case, theprotease sequestrant or inhibitor may be linked to the cellulosepolysaccharide chain through a chemical substituent such as amino,carboxylate, citrate, phosphate, sulfonate, chloride, bromide,mono-carboxylic acid, di-carboxylic acid, tri-carboxylic acid; or, anypharmaceutically acceptable salt thereof. Exemplary salts are seen toinclude those of acids such as acetic, glycolic, lactic, pyruvic,malonic, succinic, glutaric, fumaric, malic, tartaric, ascorbic, maleic,hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic,salicylic, and 2-phenoxyhenzoic; and sulfonic acids such as methanesulfonic acid and hydroxyethane sulfonic acid. Salts of the carboxyterminal amino acid moiety may include the nontoxic carboxylic acidsalts formed with any suitable inorganic or organic bases.illustratively, these salts include those of alkali metals, as forexample, sodium and potassium; alkaline earth metals, such as calciumand magnesium; light metals of Group IIA elements including aluminum,and organic primary, secondary, and tertiary amines, as for example,trialkylamines, including triethylamine, procaine, dibenzylamine,1-ethenamine, N,N′-dibenzylethylenediamine, dihydroabietylamine,N-alkylpiperidine and any other suitable amine.

The wound dressings of the instant invention may be used alone or as anadjunct to other therapeutic measures. For example, the wound dressingsmay be used together with the administration of exogenous growthfactors. Obviously, conditions that increase the stability of anexogenous peptide growth factor or its receptor will likely promote itsefficacy. The wound dressings of the present invention may also be usedin conjunction with skin grafts, in which case a proteolytic environmentthat is under control will less likely cause the “rejection” or meltingof a skin substitute graft.

The wound dressings may also include other therapeutically beneficialsubstances such as antibiotics, vitamins, and the like.

The dressings and methods of the present invention may be utilized totreat any type of appropriate wound. In a preferred embodiment, thewound that is treated is a chronic, non-healing wound.

The invention is illustrated by the following Examples which areintended to be illustrative but should in no way be construed aslimiting.

Examples Carboxymethylated and Dialdehyde Cotton Gauze MethodsPreparation of Dialdehyde Cotton Gauze

Dialdehyde cotton gauze (also referred to as 2,3dialdehyde-anhydroglucos-cellulose, oxidized cellulose, oxycellulose, orperiodite-oxidized cellulose) was prepared as follows: cotton gauze (12ply-4 in.×4 in.), USP type VII, were treated under three differentreaction conditions in lots of 50 gauze sponges as follows: Treatment 1:a 0.07 M solution of sodium periodate for 1 h at 45° C. with a solutionpH of 4.2. Treatment 2: a 0.2 M solution of sodium periodate for 1.5 hat 45° C. with a solution pH of 4.5. Treatment 3: a 0.2 M solution ofsodium periodate for 3 h at 45° C. with a solution pH of 4.5. Followingthe treatment excess periodate was removed by rinsing the gauze througha screen under running tap water. Following the rinse cycle the gauzesamples were passed through a conventional ringer to remove excessmoisture. The samples were then separated and placed on a wire rack toair dry overnight. The dried gauzes were placed in Chex all II.™.instant sealing pouches (5 in.×10 in.) and sterilized with ethyleneoxide gas by Micro Test Laboratories, Agauam, Mass.

Preparation of Carboxymethylated Cotton Gauze

Carboxymethylation was completed as outlined previously (Liyanage et al,1995). A solution was made by mixing 24 parts of dichloroacetic acidwith 24 parts of water, and while cooling in an ice bath, stirring in 75parts of sodium hydroxide solution. This solution was used to pad asample of cotton gauze to a wet pickup of 135%. The wet sample was thenplaced in an oven at 100° C. and dried/cured for 10 minutes.

Determination of Dialdehyde Content and Degree of Substitution ofCarboxymethylcellulose

Previously outlined procedures were employed to determine the dialdehydecontent (Hofreiter et al. 1995) and the degree of substitution for thecarboxymethylated gauze (Reinhardt et al.)

Assay of Treated Gauze for Elastase Activity

Treated and untreated gauze samples were submerged in 1 milliliter ofbuffer containing 0.1 units/ml of human neutrophil elastase. The sampleswere allowed to incubate for one hour at room temperature, and the gauzesamples were removed and placed in a press to drain unbound buffer andenzyme. The unbound buffer and enzyme fractions were combined andassayed for elastase activity as described below.

Enzyme Assays

Enzyme assays of the solutions containing unbound human neutrophilelastase were conducted in pH 7.6 buffer composed of 0.1 M sodiumphosphate, 0.5 M NaCl, and 3.3% DMSO and subjected to spectrophotometricmeasurement of the release of p-nitroaniline at 410 nm from theenzymatic hydrolysis of MeOSuc-Ala-Ala-Pro-Val-pNA (Sigma). Thespectrophotometric kinetic assays were performed in a BioRad MicroplateReader (Hercules, Calif.) with a 96-well format. 200 microliter aliquotsof a elatase solution (0.2 units) were assayed to initiate the enzymereaction.

Results

The gauze finishes employed in this study were prepared to assess theeffect of 1) both sterilization and variation of the sodium periodatefinishing conditions on the activity of dialdehyde cotton gauze inreducing elastase activity; and 2) the effect of the degree ofsubstitution of carboxymethylated gauze in reducing elastase activity.

As shown in FIG. 1A, variation of the oxidation conditions, and hencepercent aldehyde incorporation, effects elastase-lowering activity ofthe dialdehyde cotton gauze. The results of these studies suggests thatTreatment #1 is optimal for retaining efficacy of the dialdehyde cottongauze. Prolonged exposure and higher periodate concentration, which iscorrelated with fewer dicarbonyl units in the cotton cellulose, appearsto decrease the efficacy of the gauze in reducing elastase activity insolution.

Two different degree of substitution (DS) levels of carboxy methylatedcotton cellulose were also compared. As shown in FIG. 1B, highersubstitution levels of carboxylate on cotton resulted in an increasedreduction in elastase activity in solution.

Correlation of decreased enzyme activity with number of carboxylate oraldehyde sites on cellulose observed within a narrow range of enzymerates of activity suggests that the cotton derivatized aldehyde andcarboxylates bind elastase into readily accessible binding sites in themodified cotton fiber of the gauze.

These results suggest that dialdehyde cotton gauze and carboxymethylatedgauze can be used to effect the sequestration of the protease elastasefrom solutions of the enzyme.

Oxidized, Sulfonated, and Phosphorylated Cotton Gauze DressingsSelectively Absorb Neutrophil Elasase Activity in Solution MethodsPreparation of Periodate-Oxidized, Sulfonated, and Phosphorylated Cotton

2,3 dialdehyde-anhydroglucose-cellulose (i.e. periodate oxidized)cotton. Cotton gauze (12 ply-4 in.×4 in.), USP type VII, was treated inlots of 50 gauze sponges in a 0.07 M solution of sodium periodate for 1h at 45° C. with a solution pH of 4.2. Alternatively, cotton gauze wasoxidized with 0.2M sodium metaperiodate (pH 5) at 40° C. for 3 hours.Following the treatment excess periodate was removed by rinsing thegauze through a screen under running tap water. Following the rinsecycle, the gauze were passed through a conventional ringer to removeexcess moisture. The samples were then separated and placed on a wirerack to air dry overnight. The dried gauze are placed in a Chex allII.™. instant sealing pouch (5×10 in.) and sterilized with ethyleneoxide gas by Micro Test Laboratories, Agauam, Mass.

Sulfonated cotton. The cotton gauze may be sulfonated by washing thedialdehyde oxycellulose with 5% sodium bisulfite (NaHSO₃) under pH 4.5,liquor ratio 1:60 for 3 hours. Excess sodium bisulfite may be removed byrinsing with water under running tap water. Following the rinse cyclethe gauze are passed through a conventional ringer to remove excessmoisture. The samples are then separated and placed on a wire rack toair dry overnight.

Phosphorylated cotton. Phosphorylation of cotton gauze is accomplishedby applying inorganic phosphate salt (sodium hexametaphosphate) tocotton gauze in 4-16% composition. Urea is usually included in theformulation on a 2:1 weight ratio of urea to phosphate. All formulationscontained 0.1% Triton X-100 as a wetting agent. The cotton gauze ispadded to 80-90% wet pickup and then dried at 60° C. The samples arecured at 160° C. for 7 min.

The phosphorylated and sulfonated cotton cellulose D.S. levels were0.035 and 0.011 respectively, as measured by elemental analysis.

Carboxymethylated Cotton Gauze

Carboxymethylation was completed as outlined previously (Reinhart et al.1957). A solution was made by mixing 24 parts of dichloroacetic acidwith 24 parts of water and while cooling in an ice bath stirring in 75parts of sodium hydroxide solution. This solution was used to pad asample of cotton gauze to a wet pickup of 135%. The wet sample was thenplaced in an oven at 100° C., and dried/cured for 10 minutes.

Free-Swell Absorbency and Wicking Test

A free-swell absorbency test was performed as follows: A 0.5 gram sampleof the cotton gauze was placed in 30 mL of a 0.9% by weight aqueoussaline solution and left for 5 minutes. The cotton textile was thenfiltered through a sintered Mark 1 funnel of pore size 100-160 micronsand is left for 5 minutes, or until it stops dripping. The waterfiltered through the funnel was weighed and the weight of water absorbedby the filaments is calculated by subtraction. A wicking test was madeby immersing the cotton gauze in deionized water containing foxboro reddye such that the gauze was just touching the water surface. The timerequired for the dye solution to migrate 1.5 cm on the gauze strip wasmeasured.

Patients and Wound Fluid

Informed consent was obtained for all procedures, and approval wasreceived from the Virginia Commonwealth University Committee on theConduct of Human Research, in accordance with the 1975 Declaration ofHelsinki. Fluids were harvested from a grade III trochanteric pressureulcer of a patient with spinal cord injury using a sub-atmosphericdevice (V.A.C.™, KCl, San Antonio, Tex.). Fluids were clarified bycentrifugation at 14,000 g for 15 min at 4° C. The protein concentrationwas determined with the Bio-Rad Protein assay (Richmond, Calif.) withbovine serum albumin as a quantitation standard.

Assay of Wound Fluid

The patient wound fluid was diluted (1:100; wound fluid: buffer; v:v) ata volume of 3 mL with buffer (0.1M sodium phosphate, 0.5 M NaCl, and3.3% DMSO) and incubated with weighed samples of gauze ranging from 75mg to 700 mg. The gauze samples were soaked in the wound fluid solutionsfor one hour whereupon the solutions were filtered from the gauze underpressure applied to the gauss using a Whatman Autovial (0.45 micron PFTEmembrane). Recovery of the wound fluid solution from the gauze wasjudged to be 90%. The wound fluid solution was assayed for elastaseactivity in a manner similar to the elastase enzyme assay describedbelow. Rates of substrate hydrolysis were measured on a reactionprogress curve of absorbance versus time.

Sequestration and Inhibition of Elastase Activity by Finished CottonGauze

The effect of a variety of cotton gauze finishes was tested to assessextraction of elastase from solution. Carboxymethylated, sulfonated,phosphorylated, and oxidized cotton gauze were assayed as 50 and 75milligram samples of type VII cotton gauze (used typically in patientswith chronic wounds). Treated and untreated gauze samples were submergedin 1 milliliter of buffer containing 1 unit/mL of human neutrophilelastase. The samples were allowed to incubate for one hour at roomtemperature, and each individual gauze sample was removed and placed inan Autovial press filter (Whatman,) to extract unbound buffer andenzyme. The filtered fraction of each individual sample was re-combinedwith solution not taken up by the gauze and assayed for elastaseactivity.

The modified gauze containing bound elastase was assessed forrecoverable enzyme activity by pooling gauze samples and extractingbound elastase with 20% acetic acid solution. Samples of 1-2 grams ofmodified gauze were soaked in acetic acid solutions, filtered and thesolutions lyophilized to dryness. The lyophilized pellet was resuspendedin buffer, filtered on a sintered glass filter funnel and the resultingsolution was assayed in 200 microliter aliquots. Elastase activitiesrecovered from the gauze were 43 milliunits per gram in untreated gauzeand 160 milliunits per gram from dialdehyde cotton gauze.

Enzyme Assays

Enzyme assays of the solutions containing unbound human neutrophilelastase were conducted in pH 7.6 buffer composed of 0.1M sodiumphosphate, 0.5 M NaCl, and 3.3% DMSO and subjected to spectrophotomericmeasurement of the release of p-nitroaniline at 410 nm from theenzymatic hydrolysis of N-Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitoranilide(Sigma) (Nakajima et al. 1979). The spectrophotometric kinetic assayswere performed in a Bio-Rad Microplate Reader (Hercules, Calif.) with a96-well format. Two hundred microliter aliquots of an elastase solution(0.2 units) were assayed per well, and 20 microliters of a 60 micromolarsubstrate solution was added to initate the enzyme reaction.

Inhibition of Elastase Activity with Dialdehyde Starch

Elastase activity was measured in dialdehyde starch solutions. Solutionsof dialdehyde starch (Sigma) were prepared in the buffer described aboveat concentrations of 100 to 0.1 micromolar. The dialdehyde starchsolutions were incubated with stirring in Reacti-Vials with 0.2 units/mLof elastase for an hour. The solutions were centrifuged at 1200×g forfive minutes and the supernatant was assayed for elastase activity asdescribed above.

Results

Cotton gauze was subjected to phosphorylation, oxidation, andsulfonation. The degree of substitution (D.S.) was determined by astandard degree of substitution relationship for cellulose (based on theper cent of total phosphorous and sulfur for the phosphorylated andsulfonated samples). Base titration of free carboxyls was employed todetermine D.S. levels on carboxymethylated cotton cellulose (CMC). Thephosphorylated and sulfonated cotton cellulose D.S. levels were 0.035and 0.011 respectively. This corresponds to one phosphate for every 28anhydroglucose units and one sulfate for every 91 anhydroglucose units.The degree of substitution for the dialdehyde was also 0.011 since thebisulfite addition reaction is utilized to determine D.S. levels fordialdehyde cotton. The degree of substitution for CMC was 1.4.

Effect of Modified Gauzes on Elastase Activity

Initial experiments examined the ability of the modified cottoncelluloses to absorb purified neutrophil elastase. Twenty-five, fiftyand seventy-five milligram quantities of gauze were soaked to saturationfor an hour in one milliliter of buffered solution containing 0.2 unitsof elastase. Unbound enzyme was removed by filtration followed bypressing under high pressure. The recovery of buffer from the filtrationprocess was found to be 90%.

The assessment of elastase activity in solution exposed to the treatedgauze was performed on the unbound enzyme. Acid-extractable elastaseactivity was assayed in a 96-well format usingMeOSuc-Ala-Ala-Pro-Val-pNa for substrate hydrolysis. The kinetics ofelastase activity is based on the relative initial velocity (v.sub.o)values for enzyme solutions exposed to cotton gauze. In this study 0.2units of elastase were tested per sample. Measurement of elastaseactivity remaining in solution upon treatment with the gauze wasaccomplished by monitoring the reaction rate within a thirty-minute timeframe. The reaction progress curves for the treated samples are shown inFIG. 2 a-c. A decrease in active enzyme sites is apparent from thedecreasing dose response relation of the treated gauze samples withdialdehyde, sulfonated, and phosphorylated cotton. The decreased ratereflects a decrease in units of elastase activity retained in the elutedbuffer. A plot of v_(o) values shown in FIG. 3 for the samples alsodemonstrates this dose response relationship. The plot of v_(o) valueswas within the same range for the dialdehyde, sulfonated andphosphorylated cotton. A similar decrease in velocity was demonstratedwith increasing weight of treated gauze.

The lower v_(o) values for the treated samples when compared with theuntreated cotton gauze suggests that the elastase activity is retainedin the treated cotton gauze due to selected modifications on the gauze.Retention of elastase activity in treated gauze was found to befour-fold higher than in untreated gauze.

To assess whether the dialdehyde cotton gauze may act through activesite uptake of elastase, dialdehyde starch was employed as a solublealdehydic polysaccharide that may bind elastase. The resultsdemonstrated that inhibition of elastase by dialdehyde starch isobserved within a low micromole range, which is an inhibitoryconcentration within the titer of aldehydes per gram of dialdehydecotton used in the current study. Thus, inhibition of elastase activityby a soluble form of a high molecular weight aldehydic carbohydratesuggests that the dialdehyde cotton gauze may function as a serineprotease sequestrant through active site access to elastase.

Non-specific binding of the enzyme by the dialdehyde cotton gauze is analternative explanation for elastase inhibition by dialdehyde cottongauze. Since aldehydes can form Schiff bases with protein amino groupsthe potential for Schiff base formation between the protein amino groupsof elastase and the aldehydes of dialdehyde cotton (DAG) was a concern.To mimic the effect of protein amines a high molecular weight polylysinewas employed. Polylysine is a single amino acid biopolymer containingonly epsilon amines as the side chains of the primary amino acidstructure. To test for a potential non-specific Schiff base reactioneffect between the elastase and the DAG, the dialdehyde cotton wasincubated in a polylysine solution and elastase added to the solution totest for retention of elastase-lowering activity. DAG retained itsinhibitory effect on elastase in the presence of polylysine. Based onthis result it may be inferred that proteinaceous amines do notinterfere with the observed elastase-lowering effects of the dialdehydecotton gauze.

Elastase-Lowering Activity in Wound Fluid

The dialdehyde cotton gauze (DAG) was selected for further evaluationusing human wound fluid. To assess the ability of the modified gauze tolower wound fluid-containing elastase activity in comparison tountreated gauze (UT), DAG samples and UT were placed in wound fluid in arange of 2.5 to 20 milligrams of gauze per microliter of patient woundfluid. After exposure to the DAG or UT, the solutions of chronic woundfluid were assessed for residual elastase activity using a knownelastase substrate

The results showed that the chronic wound fluid which had been exposedto DAG possessed less elastase activity than that which had been exposedto UT at each quantity of gauze tested. This suggests that more elastasehas been sequestered by DAG than by UT. Increasing the quantity of DAGresulted in a dose dependent decrease in the amount of retained elastaseactivity.

These results reflect the superior ability of the DAG samples to removeelastase activity from wound fluid as compared to untreated cottongauze. Dialdehyde cotton gauze extracted 2-5 fold more elastase activitywith increased gauze loading per volume of wound fluid when comparedwith untreated gauze.

Measurement of protein levels remaining in the wound fluid followingincubation with the gauzes was performed to compare the relative amountsof protein taken up by treated and untreated gauze. Lower levels ofprotein were found in the wound fluid soaked with DAG than with theuntreated cotton. This is consistent with the lower activity of elastasefound in the wound fluid soaked with DAG samples.

The results obtained demonstrate that dialdehyde cotton effects thesequestration of the protease elastase from wound fluid.

We have optimized phosphorylation of cellulosic materials to achieveproducts with exceptional sequestering properties for elastase and otherproteases. These optimization involved utilization variousphosphorylation reagents, achieving products with great sequesteringproperties at high product throughput and lower reagent utilization.FIGS. 4 a-d for example show phosphorylation of cotton gauze withmonoammonium phosphate (MAP) in the presence of urea (U) at differentloading at mild temperatures. In these figures the X-axis represent the% solid content of fabric before curing (MAP+U) and the Y-axis is the %elastase sequestration (using pure neutrophil elastase at 20 mu/ml andfabric to liquid ratio of 50 mg/1 ml soln). All the fabrics had beencured for 6 minutes. It is interesting to note that (FIG. 4A, B, C) wecan carry out these reactions at temperature as low as 130 C to achieveproducts with good elastase sequestering performance (ElastaseSequestering for cotton is about 25% using pure elastase). But it ismore interesting to note that silver derivatives of these phosphorylatedcotton gauzes have outstanding elastase sequestering properties (see,FIGS. 4C and D) whereas silver derivative of product formed at 80 C doesnot show this performance. Silver derivatization was achieved bybringing 1 g of phosphorylated product with 10 ml of silver nitratesolution (200 ppm silver nitrate) for about 2 minutes followed byrinsing with excess deionized (DI) water. No free silver nitrate remainson the fabric which is white. It is apparent that phosphorylation at 80C is not adequate enough to make good performing silver derivative (FIG.4E). Additional benefits of these transition metal derivatives, such assilver or copper, is that in addition to their great elastasesequestering performance they also possess antimicrobial properties.

Moreover, by incorporating alginates and starches into cellulosicproducts we have made products with high liquid sorption and liquidretention capabilities. In experiments described below we used ammoniumalginate, Protamon S obtained from FMC Biopolymer, Philadelphia Pa.Other types of alginates such as sodium alginate, potassium alginate,magnesium alginate or alginic acid could be used as well in the wounddressing applications contemplated by this invention. Alginates arebiopolymers of mannuronic (M) and glucoronic (G) acids. Alginate occursin the seaweed as a mix of calcium, magnesium, sodium and potassiumsalts. The process of manufacturing of this product from the seaweedinvolves extraction of the alginic acid from the weed followed byneutralization by different salts to prepare appropriate product.Depending on the source of the seaweed and the extraction processdifferent grades of product with different M and G ratio and sequenceand molecular weight are obtained. Depending on the molecular weight ofthe alginate, different viscosity is obtained at the same concentration.Above 2.5% wt/wt solution the ammonium alginate we used producedsolutions with viscosity over 200 centipoise at room temperature so wekept concentration below this value.

Elastase Sequestering Performance:

The elastase sequestering performance of our products was measured byplacing 50 mg of the product in 1.00 ml of Simulated Wound Fluid (SMFL).SMFL was prepared by dissolving leukocyte elastase into buffer atconcentration of 20 m. unit/ml. Included in SMFL is 0.1% bovine serumalbumin (BSA) to simulate average chronic wound. We have found that thisformulation has activity similar to “average” chronic wound and allowsus to compare performance of different wound care product to each otherand to controls. The elastase sequestering performance of the product isobtained by comparing the activity of the SMFL fluid before and afterequilibration (2 hr) of the fluid with the product.

Competitive Liquid Sorption and Retention

This procedure is to simulate the performance of products according tothe invention when they are in touch with another liquid absorber. Theseabsorptive materials retain fluid better than the standard cottondressing. Therefore there is not only absorption of fluid, but he absorbfluid is retained within the dressing. Clinically this means thatdressing could be left in the wound for longer period of time and thenormal skin adjacent to the wound would be less likely to be macerated.This procedure is to simulate fluid sorption capacity of wound careproducts according to the invention as compared to average liquidsorption capacity of cotton gauze or paper towel. The experiment iscarried out in the following manner. A piece of the product (0.1 to 1.00gram) is allowed to come in contact with water for 60 seconds. Theamount of the water used in this experiment is in excess of totalsorption capacity of the product and we keep it to 1.5 ml water per 0.1g of the product. So if the wound care product is 0.2 gram, we use 3.0ml and if 0.5 gram, we use 7.5 ml of water.

After the product has soaked the water, it is removed and placed flat on10 folds of dry paper towels allowing excess water to drain away for 90seconds. The product is then weighed accurately and the % water retainedunder this competitive condition is calculated as described below;

% Competitive Water Sorption/Retentive=100×(Wt _(90 sec) −Wt_(Original))/Wt _(Original)

Where Wt_(90 sec) is the weight of the dressing after 90 second ofcontact with the dry paper towel and Wt_(Original) is the originalweight of the dressing. This property will be referred to % waterretention throughout this patent (% WRC).

Alginate and Silver Alginate Solution Preparation Preparation of 2.2%Alginate Solution:

Dissolved 10.0 gram of ammonium alginate in 449.0 grams of water andmixed overnight to achieve uniform solution. Solutions above 3% had toohigh viscosity and were difficult to handle without heating.

Preparation of 1.1 wt % Alginate 10% Neutralized with Silver Nitrate

Alginate solution was diluted by mixing 40 gram of the 2.2% alginatesolution with 30 gram of DI water. Silver nitrate (Reagent Grade) fromBaker was dissolved in DI water (77.4 mg in 5 ml) and added drop wiseinto the alginate solution with rapid agitation. A semi-clear solutionresulted after overnight agitation. This “solution” will settle if leftstanding indicating gel formation. When we attempted to make higherconcentration solutions, insoluble gels were formed which were hard tohandle. Also when we attempted making silver alginate solutions above10% neutralization (molar), gel formation made this product difficult tohandle. Therefore a major drawback of making silver alginate and usingthem for making products is difficulty of handling gels. Despite thisdifficulty, in some applications, silver alginates may be preferred.

Highly liquid retentive dressings can be formed by coating of cotton orphosphorylate cotton with alginate and silver alginates as shown inTable 1.

TABLE 1 % Elastase Seq. Examples Description SWFL % CWR Comparative 1Cotton Gauze 16.0 100 Comparative 2 PhosCot 44.2 100 Example 1 PhosCotw/3.7% Alg 45.3 513 Example 2 PhosCot w/3.7% Silver 77.7 540 Alg (10%)Comparative 3 Cotton w/3.7% Alg 26.3 583 Example 3 Cotton w/3.7% Alg79.4 436 (10% Silv)In Table 1, PhosCot stands for phosphorylated cotton. ComparativeExample 1 shows the behavior of common cotton which has low elastasesequestering performance and also low competitive liquid retentioncapacity. PhosCot (Comparative Example 2) has significantly higherelastase sequestering property, but it has low % CWR. Cotton coated withalginate (Competitive Example 3) has high % CWR, but it has low %elastase sequestering properties. On the other hand when phosphorylatedcotton is coated with alginate (Example 1) or with silver alginate(Example 2) we have products with both good % Elastase sequestrationsand % CWR. We can also achieve good performance when coating cotton withsilver alginate (Example 3). As we have mentioned in the silver alginatesolution preparation the viscosity and gel formation is a difficultythat could be avoided by other approaches of preparing these products bydifferent processes as described below.

Desirable products (Table 2) were prepared by coating cotton (Example 4)or phosphorylated cotton (Example 5) with ammonium alginate in the firststep, and then exposing the alginate coated fabric to silver nitratesolution to convert alginate into silver alginate to get product withhigh elastase sequestering properties (% CWR was not measured for thesesamples but were high). We used 1000 ppm silver nitrate solution forthis derivatization. The advantage of this process is that one does nothave to deal with gels of silver alginate, but the disadvantage of thisprocess is that during the silver conversion a portion of the alginateis transferred into the silver nitrate solution (in form of gels) andfrom stand point of process control this type of product formation isnot as desirable as process described below.

TABLE 2 % Elastase Sequestering Fabric Process (SWFL) Example 4 CottonW/4% Alginate Then Silver Nitrate 61.2 Example 5 Phosphorylated cottonwith 4% Alginate, 59.4 then Silver NitrateIn this more preferred embodiment, the phosphorylated cotton gauze(Comparative 2) is reacted with soluble silver compounds such as silvernitrate or acetate to form silver complex with exceptional elastasesequestering capacity (Comparative 4). However the % CWR capacity ofthis product is not much different than that of cotton. When such aproduct is then coated with alginates, products with exceptionalelastase sequestering and % CWR are obtained (Examples 6 and 7).

TABLE 3 % Elastase Seq. Examples Description (SWFL) % CWR Comparative 2PhosCot 44.2 100 Comparative 4 PhosCot, 85.0 100 Silver derivative (1000ppm AgNO3) Example 6 PhosCot, 79.0 275 Silver derivative (1000 ppmAgNO3), then 4% Alginate Example 7 PhosCot, 72.5 383 Silver derivative(1000 ppm AgNO3), 6% Alginate

While phosphorylated cotton is preferred in the present invention, otherpolysaccharide derivatives may complex with silver such asphosphorylated starch, carboxymethyl cellulose, dialdehyde gauze, andsulfonated gauze, and those of skill in the art will recognize that theinvention can be practiced with polysaccharides which have the capacityto form complexes with transition metals such as silver or copper.

It is also understood by those of skill in the art that these alginatecontaining products could be cross-linked by exposure to multivalentcations such as Ca2+, Cu2+, or Al3+ without affecting their elastasesequestering properties or their % CWR capacities as shown by examplesthat follow. In these examples, after coating the fabric with alginateor silver alginate, the products were brought in contact with 200 ppmcalcium chloride (Ca) or copper sulfate (Cu) and then the performanceswere measured. As seen these products have great performances.Additionally these products having large supply of silver or copper willhave antibacterial and antifungal properties.

TABLE 4 % Elastase Seq. Examples Description (SWFL) % CWR Example 8PhosCot w/3.7% Alg, 50.1 363 then Ca Example 9 PhosCot w/3.7% Alg 56.2453 (10% Silv), then Ca Example 10 PhosCot w/3.7% Alg, 53.1 364 then CuExample 11 PhosCot w/3.7% Alg 67.1 385 (10% Silv), then CuThese products have exceptional performance as dressings for chronicwounds where management of exudates fluids is critical in treatment ofthis class of wounds.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1-6. (canceled)
 7. A method for treating a wound, comprising: contactinga wound with a wound dressing comprising a polysaccharide substrate withan alginate coating; absorbing wound fluid with said wound dressing; andsequestering elastase from said wound.
 8. The method of claim 7 whereinsaid polysaccharide substrate is phosphorylated and is complexed with atleast one transition metal.
 9. The method of claim 7 wherein saidalginate coating is mixed with at least one transition metal.